Negligible-Cost and Weekend-Free Chemically Defined Human iPSC Culture

Abstract

Human induced pluripotent stem cell (hiPSC) culture has become routine, yet the cost of pluripotent cell media, frequent medium changes, and the reproducibility of differentiation have remained restrictive. Here, we describe the formulation of a hiPSC culture medium (B8) as a result of the exhaustive optimization of medium constituents and concentrations, establishing the necessity and relative contributions of each component to the pluripotent state and cell proliferation. The reagents in B8 represent only 3% of the costs of commercial media, made possible primarily by the in-lab generation of three E. coli-expressed, codon-optimized recombinant proteins: fibroblast growth factor 2, transforming growth factor β3, and neuregulin 1. We demonstrate the derivation and culture of 34 hiPSC lines in B8 as well as the maintenance of pluripotency long term (over 100 passages). This formula also allows a weekend-free feeding schedule without sacrificing capacity for differentiation.

Keywords: FGF2; chemically defined; culture media; differentiation; human induced pluripotent stem cell; pluripotent state; weekend-free.

Figure 1

Optimization of Basic Human Pluripotent Stem Cell Medium Constituents with a Short-Term Growth Assay Results are normalized to initial medium component concentrations shown with a dark gray bar. Optimized component concentrations are shown with a diagonal hash. Optimizations were completed using hiPSC line 19c3 and a short-term 6-day growth assay. (A) Concentrations of initial medium formula components. (B) Schematic of assay growth schedule showing seeding of cells at 10,000 cells per well of a 12-well plate, the day on which medium was changed (E8Y/E8), and the use of a PrestoBlue cell viability assay on day 6 to assess final cell number. (C) Comparison of the effect on relative growth of concentrations of recombinant human insulin (n = 19). (D) Recombinant human IGF1 LR3 (n = 3). (E) L-ascorbic acid 2-phosphate (n = 19). (F) Transferrin (n = 22). (G) Sodium selenite (n = 18). (H) FGF2-K128N (n = 6). (I) TGF-β1 (n = 22). n = full independent experimental replicates, Mann-Whitney test, ^∗p ≤ 0.05, ^∗∗∗p ≤ 0.005, ^∗∗∗∗p ≤ 0.0001, n.s., not significant. The significance bar refers to the significance between the conditions at the beginning and the end of the bar.

Figure 2

Optimization of Additional Human Pluripotent Stem Cell Medium Constituents with a Short-Term Growth Assay Results are normalized to initial medium component concentrations shown with a dark gray bar. Optimized component concentrations are shown with a diagonal hash. Optimizations were completed using hiPSC line 19c3 and a short-term 6-day growth assay. (A) Comparison of the suitability of recombinant transferrin (10 μg mL⁻¹) to support clonal growth with and without ROCK1/2 inhibition using Y27632 (10 μM) during the first 24 h after passage (n = 3). (B) Comparison of the effect on relative growth of concentrations of ROCK1/2 inhibitor Y27632 only during first 24 h after passage (n = 5). (C) Comparison of the effect on relative growth of concentrations of ROCK1/2 inhibitor thiazovivin during first 24 h after passage (n = 16). (D) Comparison of the effect on relative growth of the addition of non-essential amino acids (NEAA) and chemically defined lipids (n = 5). (E) Fatty acid-free albumin (n = 4). (F) Sodium bicarbonate (n = 5). (G) pH (n = 9). (H) Osmolarity (n = 8). (I) FGF2-G3 (n = 3). n = full independent experimental replicates, Mann-Whitney test, ^∗p ≤ 0.05, ^∗∗p ≤ 0.01, ^∗∗∗p ≤ 0.005, n.s., not significant. The significance bar refers to the significance between the conditions at the beginning and the end of the bar.

Figure 3

Optimization of B8 Medium Constituents with a Long-Term Growth Assay Results are normalized to initial medium component concentrations shown with a dark gray bar. Optimized component concentrations are shown with a diagonal hash. Optimizations were completed in hiPSC line 19c3 using a long-term five-passage, 4-day growth assay. Optimized component concentrations shown with a diagonal hash. (A) Schematic of passaging schedule. (B) Comparison of the effect on relative growth of recombinant human insulin concentrations (n = 10). (C) L-ascorbic acid 2-phosphate (n = 13). (D) Recombinant transferrin (n = 10). (E) Sodium selenite (n = 5). (F) In-house made FGF2-G3 (n = 6). (G) NODAL (n = 5). (H) Activin A (n = 5). (I) In-house made TGF-β3 after 9 passages compared with commercial TGF-β1 (n = 9). (J) Addition of NRG1 to 40 ng mL⁻¹ FGF2-G3 (n = 6). (K) Final B8 formula. n = full independent experimental replicates, Mann-Whitney test, ^∗p ≤ 0.05, ^∗∗p ≤ 0.01, ^∗∗∗p ≤ 0.005, n.s., not significant. The significance bar refers to the significance between the conditions at the beginning and the end of the bar.

Figure 4

Qualification of B8 as Suitable for hiPSC Generation and Culture (A) Demonstration of the expression of markers of undifferentiated status in hiPSC line 19c3 cultured in B8 from passage (p) 21 to p131 (left) and 37 individual hiPSC lines derived in B8 from p12 to p65 (right), assessed by flow cytometry. (B) Phase-contrast images (original magnification 10×) of hiPSC line 19c3 p44 cultured in B8. Scale bar, 100 μm. (C) Expression of markers of undifferentiated status in a variety of B8-derived hiPSC lines. Scale bar, 100 μm. (D) Example G-banding karyotype analysis of four hiPSC lines derived in B8.

Figure 5

Qualification of B8 as Suitable for hiPSC Generation and Culture (A) 19c3 hiPSC growth at low seeding densities in B8 compared with E8 (n = 8). n = full experimental replicates, Mann-Whitney test, ^∗p ≤ 0.05, n.s., not significant. (B) Assessment of stimulation of phospho-ERK after media had been stored at 37°C for 2 or 7 days, comparing in-house generated FGF2-G3 (with or without 0.5 mg mL⁻¹ fatty acid-free albumin or 100 ng mL⁻¹ heparin) with a commercial FGF2 (Peprotech). hiPSCs were starved of FGF2 for 24 h and then treated with the indicated medium for 1 h before collection for western blot, except for “No starve” cells, which were maintained in B8 with FGF2-G3. Total ERK was used as a loading control.

Figure 6

Optimization of Weekend-Free Passaging Schedule that Is Still Compatible with Monolayer Differentiation (A) Establishment of an optimal 3.5-day medium change schedule. Experiments were completed with hiPSC line 19c3. Gray box represents B8T treatment, white box represents B8 treatment, length of box represents number of days of culture in that medium (n = 8). (B) Weekend-free (WF) passage and medium change schedule. Gray shaded represent days of medium change. 1:20 refers to passage split ratio. (C) Weekend-free schedule without medium change (WF no Δ). (D) Comparison of growth when using the WF and WF no Δ schedules, with or without addition of 0.5 mg mL⁻¹ fatty acid-free albumin over 25 passages. (E) Comparison with the addition of varying levels of fatty acid-free albumin (mg mL⁻¹) to a WF schedule (n = 4). (F) Cardiac differentiation efficiency when using WF schedule (n = 5). (G) Endothelial differentiation efficiency when using WF schedule (n = 6). (H) Epithelial differentiation efficiency when using WF schedule with or without addition of 0.5 mg mL⁻¹ albumin (A) (n = 7). n = full independent experimental replicates.